Acetic acid and hypochlorous acid compositions for treatment of biofilms and wound care

ABSTRACT

Disinfecting compositions containing hypochlorous acid and acetic acid are useful for treating biofilms in or on tissue, including biofilms related to wounds or other skin trauma. The compositions are useful for treating a variety of types of tissue, both on the surface on beneath the surface of tissue. Compositions are provided having various concentrations for different tissue types and infection levels. Compositions may be provided in gel form, and may include nanoparticle encapsulated molecules for controlled release.

RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application Ser. No. 62/438,198, filed Dec. 22, 2016, and U.S.Provisional Patent Application Ser. No. 62/438,204, filed Dec. 22, 2016;and this application is a continuation-in-part of U.S. patentapplication Ser. No. 15/267,220, filed Sep. 16, 2016, which is acontinuation of U.S. patent application Ser. No. 15/167,076, filed May26, 2016, which is: (1) a continuation-in-part of U.S. patentapplication Ser. No. 14/618,820, filed Feb. 10, 2015, which is acontinuation-in-part of U.S. patent application Ser. No. 13/770,738,filed Feb. 19, 2013, which claims priority to and the benefit of U.S.Provisional application Ser. No. 61/600,344, filed Feb. 17, 2012; and(2) a continuation-in-part of U.S. patent application Ser. No.14/618,799, filed Feb. 10, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/770,738, filed Feb. 19, 2013, which claimspriority to and the benefit of U.S. Provisional application Ser. No.61/600,344, filed Feb. 17, 2012.

The contents of each of the above-referenced applications areincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The invention generally relates to compositions of acetic acid andhypochlorous acid for treating biofilms, particularly biofilms on tissueand in wounds, and also other biofilm infections.

BACKGROUND

Microbial infections that produce biofilms can pose serious healthproblems. Scientists estimate that up to 80% of all infections affectingmammals are biofilm infections. Biofilms are particularly challenging inwound management. Medical professionals have long struggled to managebiofilms in wounds, including chronic wounds and wounds related toeczema. Bacteria such as S. aureus and P. aeruginosa create biofilms onand in wounds. These infections are difficult to eradicate and preventwound healing. It has been noted, for example, that 93.5% of chronic legulcers contain S. aureus and 52.2% harbor P. aeruginosa, and ulcerscontaining P. aeruginosa are characterized by larger wound sizes andslower healing rates. See Gjodsbol et al., 2006, International WoundJournal 3(3):225-31; and see also Fazli et al., 2009, Journal ofClinical Microbiology 47(12):4084-89.

Prior art disinfecting compositions have various shortcomings. Some failto penetrate deep enough into the wound to completely eradicate abiofilm that has taken hold beneath the surface. Others disperse tooquickly and fail to attack the biofilm long enough to be effective.Still others have issues with product stability, requiring on-siteproduction, which makes them more difficult to use and less widelyavailable. Other antimicrobial compositions are abrasive to tissue orotherwise incompatible with topical use.

SUMMARY

Disinfecting compositions of hypochlorous acid and acetic acid asdescribed herein are useful for treating biofilms in or on tissue,including biofilms related to wounds or other skin trauma. Wounds areoften susceptible to microbial infection, including biofilms that formon the surface and beneath the surface of a wound. Biofilms preventhealing and can lead to chronic conditions. In particular, a preferablecomposition comprises hypochlorous acid and acetic acid because aceticacid does not harm skin and does not contain side groups that interactwith hypochlorous acid. Other organic acids that have those attributesare known and are preferred compositions of the invention.

The compositions are useful for treating a variety of types of tissue,both on the surface and beneath the surface. The hypochlorous acidcomponent is effective against surface-level biofilms or biofilm justbelow the surface of a wound, while the acetic acid is able to penetratedeeper into a wound or other tissue. Various compositions are describedherein, having different concentrations of each acid component fortreatment of diverse types of tissues and wounds. Acetic acidconcentrations greater than about 0.25% are useful for penetrating woundsurfaces. Some compositions thus have acetic acid in concentrations ofabout 1.0%, about 2.0%, or higher, up to about 5.0% Likewise,hypochlorous acid is found in different concentrations in thecompositions depending on the type of tissue to be treated. For example,hypochlorous acid at 80 to 250 ppm is useful for treating a bladder,while only 5-60 ppm is needed for a dental composition. The compositionsare particularly beneficial due to the balance between the hypochlorousacid and acetic acid, which gives the compositions the dual effect ofsurface, just beneath surface and deeper sub-surface treatment ofbiofilm infected tissue. In addition, hypochlorous acid is able to actrapidly at or near the surface; whereas acetic acid takes longer time toact and therefore can act below the wound surface.

The compositions may be provided as a gel or cream, which allows longercontact time with the tissue. For wound treatment, gels and creamcompositions help to keep the wound hydrated, promoting healing.Additionally, one or both of the acid components of the composition canbe encapsulated in a nanoparticle for controlled or delayed release.

The disclosed compositions and treatment methods are effective againstbiofilms wherever they may be found, whether in a wound, on anothertissue, on non-tissue surfaces, or in pre-surgical or post-traumasettings. Application of the compositions to the site of skin traumahelps to combat microbial infection by preventing and treating biofilms.The compositions can be used for first-aid or in a surgical setting, fortreating skin prior to, during, or after an operation. Disinfecting thearea prior to surgical incision not only helps disinfect the operationarea but also lessens the likelihood of infection spreading to othertissue during surgery. Hypochlorous acid and acetic acids compositionscan be used for debridement and cleaning of damaged tissue.

The compositions described herein can be combined with variousexcipients and carriers to facilitate topical administration to theskin. Hypochlorous acid (and organic acid) products can take the form ofgels, creams, lotions, sprays, liquids, foams, powders, and otherdelivery formulations known in the art. Alternatively, the compositionscan be provided incorporated into cloth or fibrous wipes or wounddressings.

Because the disclosed compositions are capable of penetrating intotissues, they are also effective for fighting biofilms below the surfaceof the wound, in subcutaneous tissue, such as in a surgical site or adeep tissue wound.

In certain aspects, the invention includes a composition made up ofacetic acid and hypochlorous acid. The acetic acid is present inconcentrations greater than 0.1%, and preferably greater than 0.25%. Thehypochlorous acid concentration is between 10 ppm and 1000 ppm. invarious embodiments the particular concentrations of acetic acid andhypochlorous acid depend on the intended treatment area and how deepbeneath the surface of the tissue the treatment is desired.

In embodiments, the acetic acid is present in a concentration sufficientto penetrate beneath the surface of a tissue. In some embodiments,acetic acid concentration is greater than about 0.5%, and preferablygreater than about 1.0%, and in some embodiments it is 2.0% or more. Theacetic acid may be encapsulated in a nanoparticle for controlled ordelayed release. In embodiments, the hypochlorous acid is present in aconcentration sufficient to treat biofilm on and just beneath a surfaceof a wound. The hypochlorous acid concentration may be between 20 ppmand 200 ppm. The composition may further include a gel, cream, ointment,or oil.

In related aspects, the invention involves a method for treating abiofilm in or on tissue. The method includes applying to the tissue acomposition comprising acetic acid in a concentration sufficient topenetrate skin and hypochlorous acid in an amount sufficient to removebiofilm on and just beneath a surface of the tissue.

The acetic acid may be present in amounts sufficient to remove biofilmbeneath the surface of skin. The acetic acid may be present in an amountfrom about 0.1% to about 2.0%, and the hypochlorous acid may be presentin a concentration from about 10 ppm to about 1000 ppm.

In some embodiments, the tissue to be treated is bladder tissue, thehypochlorous acid concentration is about 80-250 ppm. In otherembodiments, the tissue to be treated is lung tissue, and thehypochlorous acid concentration is about 15-60 ppm. In still otherembodiments, the tissue to be treated is dental tissue, and thehypochlorous acid concentration is about 5-60 ppm, and the acetic acidconcentration is between about 0.05% and about 5.0%.

In some embodiments, the biofilm to be treated is formed at a woundsite, including the surface and beneath the surface of the wound. Theacetic acid concentration for wound treatment may be between about 0.5%and about 5.0%.

In related aspects, a method for treating biofilm in tissue involvesapplying a nanoparticle comprising acetic acid to a tissue site in whichbiofilm formation is suspected. The nanoparticle may be lipid soluble.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing an exemplary system for producinghypochlorous acid according to methods of the invention.

FIG. 2 is a schematic showing a magnified view of the mixing deviceshown in FIG. 1 .

FIG. 3 is a schematic showing an internal view of the mixing chamber ofthe mixing device.

FIG. 4 is a schematic showing a front view of the members that dividethe mixing chamber into a plurality of sub-chambers. This view shows theapertures in the members.

FIG. 5 is a schematic showing a valve configured with measuring sensorsfor switching from a waste line to a product collection line.

FIG. 6 is a schematic showing the valve in-line with the waste line andthe product collection line.

FIG. 7 is a schematic showing another exemplary system for producinghypochlorous acid according to methods of the invention. This system isconfigured for automated use with buffered deionized water. The buffercan either be included in the inflowing water or can be introducedthrough an injection port. The buffer may also be mixed during themixing process by using NaOH in NaOCl or separately injected and aceticacid or others similar acids or bases.

FIG. 8 is a graph of a calibration curve showing HOCl concentration(ppm) calculated indirectly versus conductivity.

FIG. 9 is a graph showing a spectrophotometric analysis of the producedHOCl. The gases generally produced during production of HOCl are ClO2,Cl2O and Cl2, all of which are detectable in the visible range as yellowor yellow-red. The graph shows no absorption from colored gases in theproduced HOCl.

FIG. 10 is a graph showing the amount (parts per million (ppm)) of HOClinitially produced (T=0) and its stability over time.

FIG. 11 is a graph showing how the pH of the HOCl product changed overtime.

FIG. 12 is a graph showing the oxidation and reduction (redox) of theHOCl product over time.

FIG. 13 shows an anti-microbial composition that includes an aqueoussolution of hypochlorous acid encapsulated in a nanoparticle.

FIG. 14 is an illustration of a method of making an anti-microbialcomposition that includes an aqueous solution of hypochlorous acidencapsulated in a nanoparticle.

DETAILED DESCRIPTION

Treatment of biofilms in wounds and other tissues is achieved using acomposition of acetic acid and hypochlorous acid. The acetic acidcomponent is particularly effective for penetrating into tissues, whilethe hypochlorous acid is particularly effective for treating biofilm onthe outer surface of tissue. Acetic acid can penetrate up to 2 mm ormore beneath the surface of a wound to treat otherwise difficult toreach biofilms.

Treating both the surface-level and subdermal infections provides a dualaction treatment that is particularly needed for wound care. Chronicwounds and eczema are plagued by biofilm that affects subsurface partsof the wound. These wounds frequently have S. aureus infections whichtypically exist on or near the surface and prevent the wound fromclosing and healing. Also, P. aeruginosa infection is often present,which is generally deeper, underneath the surface of the wound. When adeep infection is present, it is important to keep the wound open andhydrated while healing from the inside out, to prevent the wound fromclosing before the deeper infection is healed. Only treating thesurface-level infection causes the wound to close and trap the deeperbiofilm inside the tissue, which can lead to sepsis and othercomplications.

A frequent problem with diabetic foot ulcers, for example, is that theyclose at the surface, creating an open cavity beneath. The cavity willcontain pus as part of the immune system response, which consists ofdebris, bacteria, and white blood cells. Pus contained in a closedcompartment, particularly in a foot ulcer, can help spread the infectionand potentially cause sepsis. In an open wound properly dressed,however, the pus will be discharged into the wound bed and the dressing.The bacteria that survive the pus environment inside a closed wound canspread infection. Therefore, removing the S. aureus infection withoutfirst or simultaneously removing the P. aeruginosa infection may notcompletely eradicate biofilm infection, leading to early closure andpossibly sepsis.

Antiseptic solutions, such as the compositions provided herein, reduceinfection in the deep areas of the wound bed and allow wound healingfrom the inside out, so that the surface does not heal faster than theinner wound. Acetic acid is present in an amount sufficient to disinfecta biofilm beneath the wound bed, and hypochlorous acid is present in anamount sufficient to disinfect the surface of the wound. The compositiontherefore allows complete disinfection of the wound to prevent prematureclosing and trapping a biofilm beneath the surface of a closed wound.

The disclosed compositions are particularly effective because balancingthe concentrations of hypochlorous acid and acetic acid allows treatmentof the surface level biofilm and also the sub-surface biofilm. Theprecise balance depends on the treatment site and the amount of surfacepenetration that is desired. The hypochlorous acid can be present inabout 10 ppm up to about 500 ppm or more. Different uses and types oftissue may require higher or lower concentrations. The acetic acid maybe present at about 0.25% up to about 2.0% or more, and preferably about1.0%. By balancing the two components, the composition can have the dualeffect of treating at the surface and beneath the surface of the tissueor wound.

Compositions consistent with the present disclosure can be created for avariety of uses. For example, a composition of relatively low aceticacid (as low as about 0.05%) and about 5-60 ppm of hypochlorous acid isa useful composition as a mouthwash for combating infection in dentaltissue. The lower concentration of acetic acid is sufficient inmouthwash compositions because the microbial infection does not tend topenetrate deep within the tissue.

For wound treatment, on the other hand, a composition may include ahigher concentration of acetic acid (about 1.0%, about 2.0%, about 3.0%,about 4.0%, or about 5.0%) to more effectively treat the biofilm deepinside the tissue.

Other uses may require more or less of each component. For example, acomposition with hypochlorous acid concentration of about 80-250 ppm isuseful for flushing a bladder, a treatment that is often needed forpatients with a urinary catheter to prevent infection or blockage. Acomposition having a hypochlorous acid concentration of about 15-60 ppmis sufficient for treatment of an infected lung.

In certain embodiments, the composition is in the form of a gel, whichallows longer contact times with the wound. Rinsing with a solution maynot be sufficient, as the contact time of the antiseptic will be veryshort. In many cases, to fully remove a biofilm, the composition shouldbe in contact with it for a prolonged period of time, ranging from a fewseconds, to several minutes, to an hour or more. The composition may beprovided in a gel or cream, which resists immediate evaporation ordispersal. Gels, creams, ointments, oils, and other similar carriers fortopical administration are known in the art.

Additionally, for wound treatment, compositions in gel form have thebenefit of maintaining moisture at the site of the wound. It isimportant to keep the wound hydrated during and after treatment with thecompositions of the invention. The disclosed compositions are mostlywater (generally 95% or more), allowing the wound to remain hydratedwhile the antiseptic ingredients of the composition fight infection inthe wound and prevent new infections from taking hold. Maintaininghydration also prevents the wound from prematurely closing and trappingbiofilm inside the tissue. Acetic acid is readily formulated in a gelbecause acetic acid is not overly reactive. Other organic acids can beused as well, and those that are less reactive are desirable.

Slow-release compositions may be used as well. In some compositions,acetic acid may be encapsulated in lipid soluble nanoparticles, whichallow the acetic acid to be carried beneath the surface of a woundbefore being released from the nanoparticle. Administration withnanoparticles of different properties allows the acetic acid to bereleased slowly over time, prevent dispersal, and provide other benefitsto administration. Acetic acid is freely diffusing, water soluble, andhas a high vapor pressure. These properties add to the difficulty ofcontrolling where the acetic acid goes. Nanoparticle encapsulated aceticacid allows the composition to be more precisely controlled.Nanoparticles are described in greater detail below and shown in FIGS.13 and 14 .

In some embodiments, the composition includes some acetic acid that isfree from nanoparticles and some acetic acid that is encapsulated withinnanoparticles. Alternatively, slow-release formulations may be used ontheir own or in combination with other instant-acting formulations. Forexample, a wound may be treated with a composition of acetic acid andhypochlorous acid, and then treated with a composition of mainly aceticacid encapsulated in nanoparticles to provide ongoing release of aceticacid into the deep parts of the wound after the initial treatment.

Production of Hypochlorous Acid Compositions

The basis of compositions and methods of the invention is theprotonation of the hypochlorite ion (OCl⁻). Using HCl or acetic acid(HAc) and NaOCl as an example, the protonation is accomplished byintroducing an acid (e.g., HCl) to the solution, which results in thefollowing reaction occurring:HCl_((aq))+NaOCl_((aq))

HOCl_((aq))+NaCl_((aq)).orHAc_((aq))+NaOCl_((aq))

HOCl_((aq))+NaA_((aq)).

The hypochlorous acid in aqueous solution partially dissociates into theanion hypochlorite (OCl⁻), thus in aqueous solution there is always anequilibrium between the hypochlorous acid and the anion (OCl⁻). Thisequilibrium is pH dependent and at higher pH the anion dominates. Inaqueous solution, hypochlorous acid, is also in equilibrium with otherchlorine species, in particular chlorine gas, Cl₂, and various chlorineoxides. At acidic pH, chlorine gases become increasingly dominant whileat neutral pH the different equilibria result in a solution dominated byhypochlorous acid. Thus, it is important to control exposure to air andpH in the production of hypochlorous acid.

Additionally, the concentration of protons (H⁺) affects the stability ofthe product. The invention recognizes that the proton concentration canbe controlled by using an acid that has a lesser ability at a given pHto donate a proton (i.e., the acid can provide buffering capacity). Forexample, conducting the process with acetic acid instead of hydrochloricacid is optimal when the desired pH of the final solution isapproximately the pKa of acetic acid. This can be achieved by mixingratios in water of 250× or greater, meaning 1 part proton donor at 100%concentration (e.g., HCl or acetic acid) to 250 parts water.

In certain embodiments, methods of manufacturing HOCl involve mixingtogether in water in an air-free environment, a compound that generatesa proton (H⁺) in water and a compound that generates a hypochloriteanion (OCl⁻) in water to thereby produce air-free hypochlorous acid. Thewater may be tap water or purified water, such as water purchased from awater purification company, such as Millipore (Billerica, Mass.).Generally, the pH of the water is maintained from about 4.5 to about 9during the method, however the pH may go above and below this rangeduring the production process. Conducting methods of the invention in anair-free environment prevents the build-up of chlorine gases during theproduction process. Further, conducting methods of the invention in anair-free environment further stabilizes the produced HOCl.

Any compound that produces a hypochlorite anion (OCl⁻) in water may beused. Exemplary compounds include NaOCl and Ca(OCl)₂. In particularembodiments, the compound is NaOCl. Any compound that produces a proton(H⁺) in water may be used with methods of the invention. Exemplarycompounds are acids, such as acetic acid, HCl and H₂SO₄. In particularembodiments, the compound is HCl. In preferred embodiments, the compoundis acetic acid because it is a weaker acid with a preferred pKa to HCl,meaning, it donates fewer protons during the reaction than HCl and isable to maintain the preferred pH level better.

Mixing can be conducted in any type of vessel or chamber or fluidicsystem. In certain embodiments, a fluidic system 100 as shown in FIG. 1is used to perform methods of the invention. The system 100 includes aseries of interconnected pipes 101 a-c with a plurality of mixingdevices 102 and 103 in-line with the plurality of pipes 101 a-c. Thepipes and the mixing devices can be interconnected using seals such thatall air can be purged from the system, allowing for methods of theinvention to be performed in an air-free environment. In certainembodiments, methods of the invention are also conducted under pressure.Making HOCl in an air-free environment and under pressure allows for theproduction of HOCl that does not interact with gases in the air (e.g.,oxygen) that may destabilize the produced HOCl.

Pipes 101 a-c generally have an inner diameter that ranges from about 5mm to about 50 mm, more preferably from about 17 mm to about 21 mm. Inspecific embodiments, the pipes 101 a-c have an inner diameter of about21 mm. Pipes 101 a-c generally have a length from about 10 cm to about400 cm, more preferably from about 15 cm to about 350 cm. In certainembodiments, pipes 101 a-c have the same length. In other embodiments,pipes 101 a-c have different lengths. In specific embodiments, pipe 101a has a length of about 105 cm, pipe 101 b has a length of about 40 cm,and pipe 101 c has a length of about 200 cm.

The pipes and mixers can be made from any inert material such thatmaterial from the pipes and mixers does not become involved with thereaction occurring within the fluidic system. Exemplary materialsinclude PVC-U. Pipes are commercially available from Georg Ficher AB.The pipes and mixers can be configured to have a linear arrangement suchthat the pipes and the mixers are arranged in a straight line.Alternatively, the pipes and mixers can have a non-linear arrangement,such that the water must flow through bends and curves throughout theprocess. System 100 shows a non-linear configuration of the pipes 101a-c and mixers 102 and 103.

Pipe 101 a is an inlet pipe that receives the water that will flowthrough the system. Generally, the water in pipes 101 a-c is under apressure of at least about 0.1 bar, such as for example, 0.2 bar orgreater, 0.3 bar or greater, 0.4 bar or greater, 0.5 bar or greater, 0.7bar or greater, 0.9 bar or greater, 1.0 bar or greater, 1.2 bar orgreater, 1.3 bar or greater, or 1.5 bar or greater. At such pressures, aturbulent water flow is produced, thus the reagents are introduced to ahighly turbulent water flow which facilitates an initial mixing of thereagents with the water prior to further mixing in the mixing devices102 and 103.

In order to control the pH during the production process, the incomingwater should have a buffering capacity in the range of pH 3.5-9.0, morepreferably from 6.0 and 8.0, to facilitate addition of the compoundsthat generates the proton and the compound that generates thehypochlorite anion. The dissolved salts and other molecules found inmost tap waters gives the tap water a buffering capacity in the range ofpH 5.5-9.0, and thus tap water is a suitable water to be used withmethods of the invention.

In certain embodiments, deionized water with the addition of knownbuffering agents to produce a water having a buffering capacity in therange of pH 3.5-9.0 is used. On example of a buffer in this particularrange is phosphate buffer. For greater process control and consistency,using a formulated deionized water may be preferable to using tap waterbecause tap water can change between locations and also over time.Additionally, using deionized water with known additives also ensures astable pH of the incoming water flow. This process is discussed ingreater detail below.

In particular embodiments, an initial pH of the water prior to additionof either the compounds that generates the proton or the compound thatgenerates the hypochlorite anion is at least about 8.0, including 8.1 orgreater, 8.2 or greater, 8.3 or greater, 8.4 or greater, 8.5 or greater,8.6 or greater, 8.7 or greater, 8.8 or greater, 8.9 or greater, 9.0 orgreater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 10.8 orgreater. In specific embodiments, the pH of the water prior to additionof either the compound that generates the proton or the compound thatgenerates the hypochlorite anion is 8.4.

Methods of making HOCl include introducing to the water the compoundthat generates the proton and the compound that generates thehypochlorite anion in any order (e.g., simultaneously or sequentially)and in any manner (aqueous form, solid form, etc.). For example, thecompound that generates the proton and the compound that generates thehypochlorite anion are each aqueous solutions and are introduced to thewater sequentially, e.g., the compound that generates the proton may beintroduced to the water first and the compound that generates thehypochlorite anion may be introduced to the water second.

System 100 is configured for sequential introduction of reagents to thewater flow, and the process is described herein in which the compoundthat generates the proton is introduced to the water first and thecompound that generates the hypochlorite anion is introduced to thewater second. In certain embodiments, the compound that generates theproton and the compound that generates the hypochlorite anion areintroduced to the water in small aliquots, e.g, from about 0.1 mL toabout 0.6 mL. The iterative and minute titrations make it possible tocontrol the pH in spite of additions of acid (compound that generatesthe proton) and alkali (the compound that generates the hypochloriteanion). In certain embodiments, no more than about 0.6 mL amount ofcompound that generates the proton is introduced to the water at asingle point in time. In other embodiments, no more than about 0.6 mLamount of the compound that generates the hypochlorite anion isintroduced to the water at a single point in time.

To introduce the reagents to the water, pipe 101 a includes an injectionport 104 and pipe 101 b includes an injection port 105. The injectionports 104 and 105 allow for the introduction of reagents to the waterflow. In this embodiments, aqueous compound that generates the proton isintroduced to the water in pipe 101 a via injection port 104. Thecompound that generates the proton is introduced by an infusion pumpthat is sealably connected to port 104. In this manner, the flow rate,and thus the amount, of compound that generates the proton introduced tothe water at any given time is controlled. The infusion pump can becontrolled automatically or manually. The rate of introduction of thecompound that generates the proton to the water is based upon theincoming water quality (conductivity and pH level) and the pressure andthe flow of the incoming water. In certain embodiments, the pump isconfigured to introduce about 6.5 liters per hour of hydrochloric acidinto the water. The introducing can be a continuous infusion or in anintermittent manner. Since the water is flowing though the pipes in aturbulent manner, there is an initial mixing of the compound thatgenerates the proton with the water upon introduction of thehydrochloric acid to the water.

Further mixing occurs when the water enters the first mixing device 102.FIG. 2 shows a magnified view of the mixing device 102 shown in FIG. 1 .In the illustrated embodiment, the mixing device includes a length ofabout 5.5 cm and a diameter of about 5 cm. One of skill in the art willrecognize that these are exemplary dimensions and methods of theinvention can be conducted with mixing devices having differentdimensions than the exemplified dimensions. Mixing device 102 includes afluidic inlet 106 that sealably couples to pipe 101 a and a fluidicoutlet 107 that sealably couples to pipe 101 b. In this manner, watercan enter the mixing chamber 108 of device 102 from pipe 101 a and exitthe chamber 108 of device 102 through pipe 101 b.

The mixing device 102 is configured to produce a plurality of fluidicvortexes within the device. An exemplary device configured in such amanner is shown in FIG. 3 , which is a figure providing an internal viewof the chamber 108 of device 102. The chamber 108 includes a pluralityof members 109, the members being spaced apart and fixed within thechamber 108 perpendicular to the inlet and the outlet in order to form aplurality of sub-chambers 110. Each member 109 includes at least oneaperture 111 that allows fluid to flow there through. FIG. 4 shows afront view of the members 109 so that apertures 111 can be seen. Thesize of the apertures will depend on the flow of water and the pressurein the system.

Any number of members 109 may be fixed in the chamber 108, the number ofmembers 109 fixed in the chamber 108 will depend on the amount of mixingdesired. FIG. 4 shows four members 109 a-d that are fixed in the chamberto produce four sub-chambers 110 a-d. The members 109 may be spacedapart a uniform distance within the chamber 108, producing sub-chambers110 of uniform size. Alternatively, the members 109 may be spaced apartat different distances within the chamber 108, producing sub-chambers110 of different size. The members 109 are of a size such that they maybe fixed to an interior wall within the chamber 108. In this manner,water cannot flow around the members and can only pass through theapertures 111 in each member 109 to move through mixing device 102.Generally, the members will have a diameter from about 1 cm to about 10cm. In specific embodiments, the members have a diameter of about 3.5cm.

A fluidic vortex is produced within each sub-chamber 110 a-d. Thevortices result from flow of the water through the apertures 111 in eachmember 109. Methods of the invention allow for any arrangement of theapertures 111 about each member 109. FIG. 4 illustrates non-limitingexamples of different arrangements of the apertures 111 within a member109. The apertures may be of any shape. FIG. 4 illustrates circularapertures 111. In certain embodiments, all of the apertures 111 arelocated within the same place of the members 109. In other embodiments,the apertures 111 are located within different places of the members109. Within a single member 109, all of the apertures 111 may have thesame diameter. Alternatively, within a single member 110, at least twoof the apertures 111 have different sizes. In other embodiments, all ofthe apertures 111 within a single member 110 have different sizes.

In certain embodiments, apertures 111 in a member 110 have a first sizeand apertures 111 in a different member 110 have a different secondsize. In other embodiments, apertures 111 in at least two differentmembers 110 have the same size. The size of the apertures will depend onthe flow of water and the pressure in the system. Exemplary aperturediameters are from about 1 mm to about 1 cm. In specific embodiments,the apertures have a diameter of about 6 mm.

The solution enters mixing device 102 through inlet 106, which issealably mated with pipe 101 a. The solution enters the chamber 108 andturbulent mixing occurs in each of sub-chambers 110 a-d as the solutionpass through members 109 a-d via the apertures 111 in each member 109a-d. After mixing in the final sub-chamber 110 d, the water exits thechamber 108 via the fluidic outlet 107 which is sealably mated to pipe101 b.

The compound that generates the hypochlorite anion is next introduced tothe solution that is flowing through pipe 101 b via injection port 105.The compound that generates the hypochlorite anion is introduced by aninfusion pump that is sealably connected to port 105. In this manner,the flow rate, and thus the amount, of compound that generates thehypochlorite anion introduced to the water at any given time iscontrolled. The infusion pump can be controlled automatically ormanually. The rate of introduction of the compound that generates thehypochlorite anion to the water is based upon properties of the solution(conductivity and pH level) and the pressure and the flow of thesolution. In certain embodiments, the pump is configured to introduceabout 6.5 liters per hour of compound that generates the hypochloriteanion into the solution. The introducing can be a continuous infusion orin an intermittent manner. Since the solution is flowing though thepipes in a turbulent manner, there is an initial mixing of the compoundthat generates the hypochlorite anion with the solution uponintroduction of the compound that generates the hypochlorite anion tothe solution.

Further mixing occurs when the solution enters the second mixing device103. Mixing device 103 includes all of the features discussed above withrespect to mixing device 102. Mixing device 103 may be configured thesame or differently than mixing device 102, e.g., same or differentnumber of sub-chambers, same or different diameter of apertures, same ordifferent sizes of sub-chambers, etc. However, like mixing device 102,mixing device 103 is configured to produce a fluidic vortex within eachsub-chamber.

The solution enters mixing device 103 through an inlet in the device,which is sealably mated with pipe 101 b. The solution enters the mixingchamber and turbulent mixing occurs in each sub-chamber of the mixingdevice as the solution pass through members in the chamber via theapertures in each member. After mixing in the final sub-chamber, thewater exits the chamber via the fluidic outlet in the mixing devicewhich is sealably mated to pipe 101 c.

At this point, the reaction has been completed and the HOCl has beenformed. The production is controlled in-line by measuring pH andconductivity. The pH is used in combination with conductivity based on apre-calibrated relation between the conductivity and concentration ofHOCl measured with spectrophotometry. The measured conductivity is ameasure of the solvent's ability to conduct an electric current.Comparing the same matrix with different known concentrations of HOCland OCl—, a calibration curve (FIG. 8 ) has been established that isused in combination with the pH meter to regulate the titrations andcontrol the process.

Pipe 101 c can be connected to a switch valve 112 that switches betweena waste line 113 and a product collection line 114. Shown in FIGS. 5 and6 . The valve 112 includes the pH meter and the conductivity measuringdevice. These devices measure the concentration (ppm), purity, and pH ofthe HOCl being produced and provide feedback for altering suchproperties of the produced HOCl. Once the HOCl being produced in pipe101 c meets the required concentration, purity, and pH, the valve 112switches from the waste line 113 to the product collection line 114 tocollect the desired product.

The HOCl that has been produced in an air-free manner is collected andbottled in an air-free manner. Placing liquids into a bottle in anair-free manner is known in the art. An exemplary method includesplacing an inflatable vessel (such as a balloon) into a bottle. Theinflatable vessel is connected directly to the collection line 114 andthe HOCl is pumped directed into the inflatable vessel in the bottlewithout ever being exposed to air. Another method involves filling thebottles under vacuum. Another air-free filling method involves fillingthe bottles in an environment of an inert gas that does not interactwith the HOCl, such as an argon environment.

The produced hypochlorous acid is air-free and will have a pH from about4.5 to about 7.5. However, the pH of the produced HOCl can be adjustedpost production process by adding either acid (e.g., HCl) or alkali(e.g., NaOCl) to the produced hypochlorous acid. For example, a pH ofbetween about 4.5 and about 7 is particularly suitable for theapplication of reprocessing heat sensitive medical instruments. Otherapplications, such as its use in non-medical environments, for exampleas in the processing of poultry and fish and general agricultural andpetrochemical uses, the breaking down of bacterial biofilm and watertreatment, may demand different pH levels.

The process can be performed manually or in an automated manner. Fluidicsystems described herein can be operably connected to a computer thatcontrols the production process. The computer may be a PCL-logiccontroller system. The computer opens and closes the valves for thewater inlet, the waste water outlet, and the product outlet according tothe feedback received from the sensors in the system (e.g.,conductivity, pH, and concentration of product (ppm) being produced).The computer can also store the values for the water pressures and wateramounts and can adjust these according to the feedback received from thesensors regarding the properties of the HOCl being produced. Thecomputer can also control the infusion pumps that inject the reagentsinto the water for the production process.

The process can be performed iteratively in that pipe 101 c can beattached to a second fluidic system and the produced HOCl is then flowedthrough the second system where the process described above is repeatedwith the starting solution being HOCl instead of water. In this manner,an increased yield of HOCl is produced. Any number of fluidic systemsmay be interconnected with methods of the invention.

FIG. 7 is a schematic showing another exemplary system 200 for producinghypochlorous acid according to methods of the invention. System 200 isconfigured for regulation of the pH of the incoming water and injectingbuffer for stability. In system 200, water is introduced into pipe 201a. Pipe 201 a has a pH meter 208 connected to it. pH meter 208 measuresthe pH of the incoming water. The pH meter 208 is connected to injectionport 202. The injection port 202 allows for the introduction of at leastone buffering agent to the incoming water. The buffering agent isintroduced by an infusion pump that is sealably connected to port 202.In this manner, the flow rate, and thus the amount, of buffering agentintroduced to the water at any given time is controlled. The infusionpump can be controlled automatically or manually. The rate ofintroduction of the buffering agent to the water is based upon theincoming water quality (conductivity and pH level), the buffercomposition, and the pressure and the flow of the incoming water. Theintroducing can be a continuous infusion or in an intermittent manner.Since the water is flowing through the pipe 201 a in a turbulent manner,there is an initial mixing of the buffering agent with the water uponintroduction of the buffering to the water. This initial mixing may besufficient to properly adjust the properties of the incoming water.

In certain embodiments, further mixing of the water and buffer isperformed prior to conducting the process of producing the HOCl. Inthose embodiments, further mixing occurs when the water with bufferingagent enters the first mixing device 203. Mixing device 203 includes allof the features discussed above with respect to mixing device 102.Mixing device 203 may be configured the same or differently than mixingdevice 102, e.g., same or different number of sub-chambers, same ordifferent diameter of apertures, same or different sizes ofsub-chambers, etc. However, like mixing device 102, mixing device 203 isconfigured to produce a fluidic vortex within each sub-chamber.

The solution enters mixing device 203 through an inlet in the device,which is sealably mated with pipe 201 a. The solution enters the mixingchamber and turbulent mixing occurs in each sub-chambers of the mixingdevice as the solution pass through members in the chamber via theapertures in each member. After mixing in the final sub-chamber, thewater exits the chamber via the fluidic outlet in the mixing devicewhich is sealably mated to pipe 202 b. The water has a pH of at leastabout 8.0, preferably 8.4, and a buffering capacity of pH 5.5-9.0.

The process is now conducted as described above for producing HOCl. Thecompound that generates the proton is next introduced to the water thatis flowing through pipe 201 b via injection port 204. The compound thatgenerates the proton is introduced by an infusion pump that is sealablyconnected to port 204. In this manner, the flow rate, and thus theamount, of compound that generates the proton introduced to the water atany given time is controlled. The infusion pump can be controlledautomatically or manually. The rate of introduction of the compound thatgenerates the proton to the water is based upon properties of the water(conductivity and pH level), the buffer composition, and the pressureand the flow of the water. In certain embodiments, the pump isconfigured to introduce from about 6.5 liters per hour to about 12liters per hour of compound that generates the proton into the water.The introducing can be a continuous infusion or in an intermittentmanner. Since the water is flowing though the pipes in a turbulentmanner, there is an initial mixing of the compound that generates theproton with the water upon introduction of the hydrochloric acid to thewater.

Further mixing occurs when the solution enters the second mixing device205. Mixing device 205 includes all of the features discussed above withrespect to mixing device 102. Mixing device 205 may be configured thesame or differently than mixing device 203, e.g., same or differentnumber of sub-chambers, same or different diameter of apertures, same ordifferent sizes of sub-chambers, etc. However, like mixing device 203,mixing device 205 is configured to produce a fluidic vortex within eachsub-chamber.

The solution enters mixing device 205 through an inlet in the device,which is sealably mated with pipe 201 b. The solution enters the mixingchamber and turbulent mixing occurs in each sub-chambers of the mixingdevice as the solution pass through members in the chamber via theapertures in each member. After mixing in the final sub-chamber, thewater exits the chamber via the fluidic outlet in the mixing devicewhich is sealably mated to pipe 201 c.

The compound that generates the hypochlorite anion is next introduced tothe solution that is flowing through pipe 201 c via injection port 206.The compound that generates the hypochlorite anion is introduced by aninfusion pump that is sealably connected to port 206. In this manner,the flow rate, and thus the amount, of compound that generates thehypochlorite anion introduced to the water at any given time iscontrolled. The infusion pump can be controlled automatically ormanually. The rate of introduction of the compound that generates thehypochlorite anion to the water is based upon properties of the solution(conductivity and pH level) and the pressure and the flow of thesolution. In certain embodiments, the pump is configured to introduceabout 6.5-12 liters per hour of compound that generates the hypochloriteanion into the solution. The amount introduced depends on the desiredconcentration of HOCl (ppm) and flow of water through the pipes. Theintroducing can be a continuous infusion or in an intermittent manner.Since the solution is flowing though the pipes in a turbulent manner,there is an initial mixing of the compound that generates thehypochlorite anion with the solution upon introduction of the compoundthat generates the hypochlorite anion to the solution.

Further mixing occurs when the solution enters the second mixing device207. Mixing device 207 includes all of the features discussed above withrespect to mixing device 102. Mixing device 207 may be configured thesame or differently than mixing devices 205 or 203, e.g., same ordifferent number of sub-chambers, same or different diameter ofapertures, same or different sizes of sub-chambers, etc. However, likemixing devices 205 and 203, mixing device 207 is configured to produce afluidic vortex within each sub-chamber.

The solution enters mixing device 207 through an inlet in the device,which is sealably mated with pipe 201 c. The solution enters the mixingchamber and turbulent mixing occurs in each sub-chambers of the mixingdevice as the solution pass through members in the chamber via theapertures in each member. After mixing in the final sub-chamber, thewater exits the chamber via the fluidic outlet in the mixing devicewhich is sealably mated to pipe 201 d.

At this point, the reaction has been completed and the HOCl has beenformed. The produced HOCl can be measured and collected as describedabove. Pipe 201 d can be connected to a switch valve that switchesbetween a waste line and a product collection line. The valve includes apH meter and a conductivity measuring device. These devices measure theconcentration, purity, and pH of the HOCl being produced and providefeedback for altering such properties of the produced HOCl. Once theHOCl being produced in pipe 201 d meets the required concentration,purity, and pH, the valve switches from the waste line to the productcollection line to collect the desired product.

In another embodiment, a deionizer is placed in-line with incomingwater. The deionizer deionizes the water and then a buffering agent isadded to the deionized water. The production process is then conductedas described for embodiments of system 200 to produce water having a pHof at least about 8, for example 8.4, and a buffering capacity of pH6-8.

The HOCl produced by the above process can be used in numerous differentapplications, for example medical, foodservice, food retail,agricultural, wound care, laboratory, hospitality, dental,delignification, or floral industries.

Wound Care

In certain embodiments, compositions of the invention are used for woundcare. Wound care involves treating damaged or broken skin, includingabrasions, lacerations, ruptures, punctures, or burns. Particular woundcare treatments involve treating biofilms. Biofilms may form when freefloating microorganisms such as bacteria and fungus attach themselves toa surface. Biofilms are known to impair cutaneous wound healing andreduce topical antibacterial efficiency in healing or treating infectedwounds. Other common health conditions related to biofilms includeurinary tract infections, middle-ear infections, chronic wounds, and theformation of dental plaque. Cystic fibrosis, native valve endocarditis,otitis media, periodontitis, and chronic prostatitis also involvemicroorganisms that produce biofilms. Microorganisms commonly associatedwith biofilms include Candida albicans, coagulase-negativestaphylococci, Enterococcus, Klebsiella pneumoniae, Pseudomonasaeruginosa, Staphylococcus aureus, and others.

Biofilms are often resistant to traditional antimicrobial treatments,and are therefore a serious health risk. The resistance of biofilmsrenders traditional antibiotic and antimicrobial treatments ineffective.Because biofilms can greatly reduce susceptibility to antibiotics anddisinfectants, treatments are needed that are capable of breaking downbiofilms but that are not too toxic to the patient.

Methods are provided for administration of a composition to anindividual in need of treatment for a biofilm-associated infection.Methods of the invention include prophylaxis, therapy, or cure of abiofilm-associated infection. Methods include administration of one ormore unit doses of a composition in a therapeutically orprophylactically effective amount for treatment of an existingbiofilm-associated infection or prevention of establishment of abiofilm-associated infection in the individual. In some embodiments,spread of a biofilm-associated infection to another site in theindividual is inhibited. In various embodiments, the composition may beadministered parenterally, orally, locally, or topically. Compositionsmay be applied by intravenous, intra-muscular, or subcutaneousinjection. In methods of the invention, compositions may be administeredin a pharmaceutically acceptable carrier, examples of which arediscussed below.

Treatment includes killing of microbes inhabiting the biofilm orremoving a biofilm, inhibiting biofilm formation, and disrupting anexisting biofilm. The compositions disclosed herein are particularlyeffective for treatment of microbial biofilms in or on a wound. Thecomposition may be in the form of a topically administrable woundtreatment composition which comprises a hypochlorous acid and aceticacid compound. The composition may be combined with an additionalantimicrobial agent.

Compositions of the invention can be administered topically to asubject, e.g., by the direct laying on or spreading of the compositionon the epidermal or epithelial tissue of the subject. The compositionmay be formulated as a liquid, powder, lotion, cream, gel, oil,ointment, gel, solid, semi-solid formulation, or aerosol spray. Suchformulations may be produced in a conventional manner using appropriatecarriers which are well known to a person skilled in the art.

Suitable carriers for topical administration preferably remain in placeon the skin as a continuous film, and resist being removed byperspiration or immersion in water. The carrier may includepharmaceutically-acceptable emollients, emulsifiers, thickening agents,solvents, and the like.

The composition may be provided as part of a wound dressing in which thecomposition is provided within the wound dressing or on thewound-contacting surface thereof. A wound dressing may be intended to beapplied to a wound to be treated and which comprises a substratecomprising compositions in accordance with the invention. Such adressing is particularly convenient because it delivers the compositionof the invention to the wound to be treated and simultaneously providesa dressing therefor. The wound dressing may, for example, be fibrous, afoam, a hydrocolloid, a collagen, a film, a sheet hydrogel or acombination thereof. The wound dressing may be in the form of a layereddressing in which one or more layers of the dressing are formed at leastin part or one or of; natural fibers, alginate, Chitosan, Chitosanderivatives, cellulose, carboxymethyl-cellulose, cotton, Rayon, Nylon,acrylic, polyester, polyurethane foam, hydrogels, hydrocolloids,polyvinyl alcohol, starch, a starch film, collagen, hylaronic acid andits derivatives, biodegradable materials, and other materials known inthe art. Methods of the invention may further comprise negative-pressurewound therapy, as is known in the art. Such therapies involve applyingnegative pressure to the wound, such as with a vacuum dressing.

The composition may be administered in a single daily dose or inmultiple doses, e.g., 2, 3, 4, or more doses, per day. The total dailyamount of composition may be about 0.01 mg, 0.1 mg, 1 mg, 2 mg, etc., upto about 1000 mg. In some embodiments, the total daily amount ofadministered is about 0.01 mg to about 1 mg, about 1 mg to about 10 mg,about 10 mg to about 100 mg, about 100 mg to about 500 mg, or about 500mg to about 1000 mg. The actual dosage may vary depending upon thespecific composition administered, the mode of administration, the typeor location of biofilm to be treated, and other factors known in theart. In some embodiments a dosage can also be selected so as to providea predetermined amount of composition per kilogram of patient weight.

The use of the compound in conjunction with another known antimicrobialtreatment may increase the efficacy of the antimicrobial agent. In someembodiments, methods of the invention further comprise administration(simultaneously or sequentially with compositions of the invention) ofone or more doses of an antibiotic substance, including, but not limitedto, ciproflaxin, ampicillin, azithromycin, cephalosporin, doxycycline,fusidic acid, gentamycin, linezolid, levofloxacin, norfloxacin,ofloxacin, rifampin, tetracycline, tobramycin, vancomycin, amikacin,deftazidime, cefepime, trimethoprim/sulfamethoxazole,piperacillin/tazobactam, aztreanam, meropenem, colistin, orchloramphenicol. In some embodiments, methods of the invention furthercomprise administration of one or more doses of an antibiotic substancefrom an antibiotic class including, but not limited to, aminoglycosides,carbacephem, carbapenems, first generation cephalosporins, secondgeneratin cephalosporins, third generation cephalosporins, fourthgeneration cephalosporins, glycopeptides, macrolides, monobactam,penicillins, polypeptides, quinolones, sulfonamides, tetracyclines,lincosamides, and oxazolidinones. In some embodiments, methods of theinvention comprise administration of a nonantibiotic antimicrobialsubstance, including but not limited to sertraline, racemic andstereoisomeric forms of thioridazine, benzoyl peroxide, taurolidine, andhexitidine.

Treating Biofilm on Other Tissues

Compositions of the invention can be used to treat biofilms affectingvarious parts of the body, or attached to various surfaces. In someembodiments, methods of the invention comprise administration of atherapeutically effective composition to an individual in need thereoffor treatment of a biofilm-associated infection in the bladder, kidney,heart, middle ear, sinuses, skin, lung, a joint, subcutaneous tissue,soft tissue, vascular tissue, and/or the eye. In other embodiments, atherapeutically effective amount of composition is administered to anindividual in need thereof for treatment of one or more of the followingconditions associated with biofilm: urinary tract infection; chronicbacterial vaginosis; prostatitis; bacterial infection stemming fromdiabetes, such as a diabetic skin ulcer; pressure ulcer; venouscatheter-associated ulcer; or a surgical wound (e.g., a surgical siteinfection). In some embodiments, the biofilm is on the skin of anindividual. In some embodiments, the biofilm is associated with a wound,including abrasions, lacerations, ruptures, punctures, burns, andchronic wounds. In some embodiments, the biofilm is below the surface ofthe skin, in subcutaneous tissue, such as a deep tissue wound or asurgical site infection.

Treating Biofilm on Non-Tissue Surfaces

Other applications for treating biofilms are also envisaged. Forexample, the composition of the invention has application for thetreatment of microbial biofilms on surfaces, e.g. surfaces in hospitals(such as operating rooms or patient care rooms) as well as othersurfaces (e.g., household work surfaces). The invention also encompassestreating biofilms that form on implanted medical devices andprosthetics.

As is known in the art, implanted medical devices are susceptible tobiofilm formation, including fungal biofilms and bacterial biofilms.Methods and compositions of the invention can also be used to treatbiofilms that form on the surfaces of implanted medical devices such ascatheters and prosthetics. Compositions of the invention can be appliedto a medical device pre-implantation. Alternatively, the medical devicecan comprise a reservoir containing the composition, such that thecomposition can be released in a controlled manner after implantation.Methods for treating implanted medical devices can be found in U.S. Pat.Nos. 5,902,283 and 6,589,591, and U.S. Patent Publication 2005/0267543,each of which is incorporated by reference herein in its entirety.

Dental Treatment

In another embodiment of the invention, a method is provided fortreating an orally-associated biofilm such as dental plaque. Theinvention provides methods for oral plaque prevention, treating oralplaque infection, treating tooth hypersensitivity, sterilizing a rootcanal, or treating a dental disease.

Methods of the invention comprise contacting an oral surface, such asteeth, gums, gingiva, or tongue, with a therapeutically effective amountof the composition. Some methods of the invention comprise prevention ofan orally-associated biofilm by administration of a prophylacticallyeffective amount of composition to an individual. The composition may beformulated as a dentrifice, such as toothpaste, for treatment orprevention of dental plaque. In other embodiments, the biofilm may belocated on the tongue, oral mucosa, or gums. In some embodiments, thecomposition is formulated as a mouthwash. In some embodiments, thecomposition is formulated as a paint, foam, gel, or varnish, forexample, in a fluoride-containing composition. In an embodiment, thecomposition is in the form or a gel or foam in a mouthguard that apatient wears for several minutes for fluoride treatment. In otherembodiments the composition is contacted to an adhesive strips, whichcan be applied to the teeth or other oral surface. The composition maycomprise a liquid polymer formulation, which is a composition that ispreferably topically applied to a surface such as a tooth, to skin, to amucous membrane, and which dries as a film adhering to that surface, ina manner which resists removal under normal conditions, such as eatingor brushing, for applications to the teeth and oral mucosa, or normalwashing and abrasion, when applied to skin. Alternatively, thecomposition may be applied to bandages, dressings, gauze, brushes,implants, etc. and permitted to dry into a film in advance of itsadministration to a patient.

Mastitis Treatment

In another embodiment of the invention, compositions and methods areprovided for treating mastitis. Mastitis is an inflammation tissue inthe breast or udder of a mammal. It is often associated with bacterialinfections such as Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus epidermidis, Streptococcus agalactiae, Streptococusuberis, and others. Some of the bacteria known to cause mastitis alsoform biofilms, but not all mastitis results from biofilm formation.

Mastitis can occur in any mammal, such as humans, cows (dairy cattle),and other animals. Mastitis is a particular problem for dairy cattle. Incattle the condition occurs when leukocytes are released into themammary gland, often as a response to bacteria in the teat canal. Cowsthat are repeatedly infected often must be culled to prevent widespreadinfection in the herd. The loss of milk from infected cows and the lossof cows and entire herds due to infection results in large economiclosses for the dairy industry worldwide. In the United States, forexample, mastitis is estimated to cost the dairy industry up to $2billion each year.

Methods are provided for administration of a composition to a mammal inneed of treatment for mastitis. Methods of the invention includeprophylaxis, therapy, or cure for mastitis. In some embodiments, spreadof mastitis to another quarter or to another animal is inhibited.

The formulations, dosages, and routes of administration discussed aboveare applicable to these embodiments of the invention. For example, invarious embodiments, the composition may be administered parenterally,orally, locally, or topically. Compositions may be applied byintravenous, intra-muscular, or subcutaneous injection. Compositions maybe applied by infusion via a teat canal, as is known in the art. Inmethods of the invention, compositions may be administered in apharmaceutically acceptable carrier, which may include emollients,emulsifiers, thickening agents, solvents, and the like.

The composition may be administered in a single daily dose or inmultiple doses, e.g., 2, 3, 4, or more doses, per day. The total dailyamount of composition may be about 0.01 mg, 0.1 mg, 1 mg, 2 mg, etc., upto about 1000 mg. In some embodiments, the total daily amount ofadministered is about 0.01 mg to about 1 mg, about 1 mg to about 10 mg,about 10 mg to about 100 mg, about 100 mg to about 500 mg, or about 500mg to about 1000 mg. The actual dosage may vary depending upon thespecific composition administered, the mode of administration, and otherfactors known in the art. The composition may be administered inconjunction with another known antimicrobial treatment such as anantibiotic.

Compositions can be administered topically to a cow's udder by directlyapplying or spreading the composition onto the udder or teat. Thecomposition may be formulated as a liquid, powder, lotion, cream, gel,oil, ointment, gel, solid, semi-solid formulation, or aerosol spray.Methods of the invention may further comprise dipping a teat into thecomposition. Teat dipping can be used to treat an already infected udderor to prophylactically prevent mastitis from developing. The compositionmay be applied immediately before milking, immediately after milking, orboth. Methods of teat dipping are known in the art, and are described inmore detail in U.S. Pat. No. 4,113,854, as well as U.S. PatentPublications 2003/0235560 and 2003/0113384, each of which isincorporated by reference herein in its entirety. Methods may furthercomprise use of a teat sealant to create a physical barrier for the teatorifice after administration of the composition.

In other embodiments, compositions can be provided via intramammaryinfusion. Intramammary infusion involves forcing the antibiotic upthrough the teat canal into the udder. Infusion liquid may comprise acomposition disclosed herein in combination with a pharmaceuticallyacceptable carrier such as canola oil. Prior to infusion, the teat iscleaned, for example with an alcohol swab. An antibiotic infusing devicemay include a cannula sized and shaped to fit into the teat canal. Thecannula may be fully or partially inserted through the streak canal.Methods for infusion are known in the art and are described, forexample, in U.S. Pat. Nos. 4,983,634 and 5,797,872, the entirety of eachof which is incorporated by reference herein.

Methods of the invention may further comprise administering antibioticsin conjunction with compositions of the invention, or in sequentialdoses before or after administration of the compositions.

Wounds and Surgical Uses

The compositions can be applied to prevent and treat biofilm on othertypes of living tissue as well. Tissue includes, for example, skin,mucus membranes, wounds, or ostomies. As has been described above, thecomposition is useful for wound treatment. Wounds include bedsores,chronic wounds, burns, pressure wounds, diabetes wounds, and other formsof skin trauma. Wounds are often susceptible to biofilm formation, whichprevents healing and can lead to chronic conditions. Hypochlorous acidand acetic acid compositions can be used for debridement and cleaning ofdamaged tissue.

The compositions can also be used in a surgical setting, for treatingskin prior to or after an operation. The composition prevents infectionthat would lead to biofilm formation. At times, an area requiringsurgery such as a traumatic wound may already be at risk of havingdeveloped a biofilm. The hypochlorous acid composition can be used todisinfect the area prior to surgical incision, which would not only helptreat the biofilm but also lessen the likelihood of it spreading toother tissue during surgery. The compositions may be used to disinfectany surface within a surgical operating field.

Other Medical Uses

In addition to wound care, HOCl compositions of the invention can alsobe used for non-traumatic tissue treatment. They may be used for bladderirrigation, for preventing or treating bladder infections orcatheter-associated urinary tract infections, and the like. They may besimilarly used to treat infections in the aerodigestive tract such assinus and lung infections, or infections in the oral cavity, pharynx,paranasal sinuses, sinonasal tract, larynx, pyriform sinus, oresophagus. They can be used to fight microbial growth that leads toinfection and to reduce allergens that cause adverse immune responses.The compositions may also be administered to the gastrointestinal tract,including the stomach, intestines, and colon, to combat microbialinfections such as gastroenteritis, Clostridium difficile infection, andsmall-intestine bacterial overgrowth (SIBO).

In various other embodiments, the compositions can be administered inthe form of eye drops to fight eye infections, or can be used to cleanor store contact lenses to prevent bacterial growth and biofilmformation. In other embodiments, the composition can be used as an oralrinse or mouthwash to fight biofilm buildup in the oral cavity, or itcan be used to clean or store dentures.

In addition to the use as an antiseptic to treat or prevent biofilms onliving tissue, the compositions can be used as a disinfectant on othersurfaces such as for use in healthcare facilities, food preparation,cooking utensils, and the like. Compositions can be used to disinfectcountertops, hospital beds, or food preparation surfaces.

The compositions can be used to disinfect medical devices and surgicalinstruments, for example. Medical devices are often initially suppliedas sterile, but may require additional or subsequent cleaning anddisinfection or sterilization. Reusable medical devices in particularmust be sterilized or disinfected prior to reuse. Compositions can beapplied to the medical device using any known technique. For example,the composition can be applied by wiping or spreading it onto thesurface of the device, by spraying an aerosol or mist form of thecomposition onto the device, by dipping the device into a vesselcontaining a volume of the composition, or by placing the device into aflow of the composition such as from a faucet. Additionally oralternatively, medical devices and surgical instruments may also bestored submerged in the composition and removed at the time of use.

Treatment with a hypochlorous acid composition disclosed herein can bedone in addition to other known techniques such as autoclaving.Alternatively, the composition can be applied instead of autoclaving.Because heat sterilization is not useful for all devices (e.g., somedevices contain delicate parts or electronics that cannot withstand hightemperatures), hypochlorous acid compositions are a useful alternative,providing an effective way to sterilize or disinfect such devices.

The compositions can also be used to disinfect implants and prosthesesbefore introducing them to the body. Such devices include orthopedicimplants, wires, screws, rods, artificial discs, prosthetic joints, softtissue fillers, pacemakers, intra-uterine devices, coronary stents, eartubes, artificial lenses, dental implants, and many others known in theart.

The stabilized hypochlorous acid and acetic acid compositions describedherein are useful for both biofilm prevention and biofilm removal on allof the surfaces and tissues discussed herein. Because biofilms makemicrobes far more resistant to traditional antimicrobial agents,microbes that form biofilms are more able to share and modify theirresistance genes and spread into the air and surroundings. As aconsequence of biofilm development, a simple infection may becomechronic, antibiotics and antiseptics stop working, and new strains ofinfections emerge.

Both acetic acid (or other organic acids) and hypochlorous acid,however, are particularly useful for treating and preventing biofilms.The HOCl compositions disclosed herein mimic the natural disinfectant ofthe immune system. Therefore the compositions are not susceptible tomicrobial resistance. Additionally, they are non-toxic, do not sting,and relieve itching.

The various embodiments and uses described above involve a variety ofmethods of administration, as would be understood in the art.

Hypochlorous acid compositions are particularly effective fortransdermal treatment due to the small size of the HOCl molecule.Hypochlorous acid is able to penetrate epithelium and wound surfaces,and so can generally reach deeper tissue layers without requiringinjection. This is particularly useful for biofilm infection that formsbeneath the top layer of skin. Unlike many other antimicrobialtreatments, acetic acid and similar organic acids can penetrate deeperlayers of skin without requiring an invasive delivery mechanism.

In some embodiments, however, it may be desirable to prevent the HOClfrom penetrating into the skin, and therefore the compositions can becombined with excipients, carriers, emulsifiers, polymers, or otheringredients, examples of which are discussed in U.S. Patent Application2016/0271171, which is incorporated herein by reference in its entirety.

In addition to topical use, wherein the compositions can be sprayed,wiped, or rubbed onto skin, in other embodiments the compositions may beinjected into a particular tissue requiring treatment. The compositionscan be ingested in capsule form for administration to thegastrointestinal tract. They can be supplied in slow-release ordelayed-release capsules. The compositions can be provided as asuppository for insertion into the rectum or vagina.

In other embodiments, the compositions can be provided as a nasal sprayfor treatment of the aerodigestive tract, which can include treatment ofallergic reactions, sinus infections and the like. The nasal spray maybe in droplet, aerosol, gel, or powder form. The buffered hypochlorousacid and acetic acid composition can be combined with one or more of adecongestant or an anti-inflammatory or anti-histamine agent, as needed.The composition can be aerosolized with a nasal spray dispenser as isknown in the art.

The nasal spray may also include a pharmaceutically acceptable carrier,such as a diluent, to facilitate delivery to the nasal mucosa. Thecarrier might be an aqueous carrier such as saline. The composition maybe isotonic, having the same osmotic pressure as blood and lacrimalfluid. Suitable non-toxic pharmaceutically acceptable carriers are knownto those skilled in the art. Various carriers may be particularly suitedto different formulations of the composition, for example whether it isto be used as drops or as a spray, a nasal suspension, a nasal ointment,a nasal gel or another nasal form. Other additives, excipients,emulsifiers, dispersing agents, buffering agents, preservatives, wettingagents, consistency aids, and gelling agents may be included as well.Preferably, additives should be chosen that impart the desiredcharacteristic without reducing the stability of the hypochlorous acid.Additives may aid in evenly administering the composition over themucosa or for reducing or delaying the rate of absorption of thecomposition.

The composition can be delivered by various devices known in the art foradministering drops, droplets, and sprays. The nasal spray compositioncan be delivered by a dropper, pipet, or dispensing. Fine droplets,sprays, and aerosols can be delivered by an intranasal pump dispenser orsqueeze bottle. The composition can also be inhaled via a metered doseinhaler, such as a dry powder inhaler or a nebulizer.

Controlled Release with Nanoparticle Encapsulation

Stable aqueous solutions of hypochlorous acid and/or acetic acid can beencapsulated in nanoparticles that allow controlled release of the acidfrom the nanoparticles. Controlled release allows persistentanti-microbial protection.

FIG. 13 shows an anti-microbial composition 1301 comprising an aqueoussolution 1303 of hypochlorous acid encapsulated in a nanoparticle 1305.The aqueous solution 1303 of hypochlorous acid is made by a methoddescribed herein to produce a solution in which the acid is stable. Thestable hypochlorous acid solution 1303 is then encapsulated innanoparticle 1305. The nanoparticle allows gradual release of thehypochlorous acid. Although not depicted, acetic acid can also beencapsulated in a nanoparticle for controlled release.

The nanoparticle may be any type of nanoparticle that providescontrolled release of the acid from the nanoparticle. The nanoparticlemay comprise a polymer, such as an organic polymer. Examples of polymerssuitable for controlled-release nanoparticles include acrylic acid,carrageenan, cellulosic polymers (e.g., ethyl cellulose or hydroxypropylcellulose), chitosan, cyclodextrins, gelatin, guar gum, high amylasestarch, hyaluronic acid, locust bean gum, pectin, polyacrylamide,poly(D,L-lactide-co-glycolide acid), poly(lactic acid), poly(xylitoladipate salicylate), polyanhydride, poly(ethylene oxide),poly(ethyleneimine), polyglycerol ester of a fatty acid,polysaccharides, polyvinyl alcohol, povidone, sodium alginate, andxanthan gum. For details on the use of polymers to formcontrolled-release nanoparticles, see Binnebose, et al., PLOS Negl TropDis 9:e0004713 (2015); Campos, et al., Scientific Reports 5:13809(2015); Dasgupta et al., Mol. Pharmaceutics 12:3479-3489; Gao, et al.,The Journal of Antibiotics 64:625-634, (2011); Lee, et al.,International Journal of Nanomedicine 11:285-297 (2016); and U.S. Pat.No. 8,449,916 (incorporated by reference). The nanoparticle may containan aluminosilicate (such as a zeolite, e.g., analcime, chabazite,clinoptilolite, heulandite, leucite, montmorillonite, natrolite,phillipsite, or stilbite), calcium ammonium nitrate, hydroxyapatite(e.g., urea-modified hydroxyapatite), metal hydroxide, metal oxide,polyphosphate, or silicon compound (e.g., silicon dioxide). Thenanoparticle may contain lipids, i.e., it may be a lipid nanoparticle.The nanoparticle may include a liposome. For details on the use ofliposomes to form controlled-release nanoparticles, see Weiniger et al.,Anaesthesia 67:906-916 (2012). The liposome may be multi-lamellar. Thenanoparticle may contain a gel, sol-gel, emulsion, colloid, or hydrogel.For details on the use of hydrogels to form controlled-releasenanoparticles, see Grijalvo et al., Biomater. Sci. 4:555 (2016). Thenanoparticle may contain a combination of formats, such as a hydrogelencapsulated within a liposome. The nanoparticle may have a core-shellstructure. The nanoparticle may be biodegradable. Compositions of theinvention may include an anti-metabolic agent. The anti-metabolic agentmay be a metal ion. For example, the anti-metabolic agent may be zinc,copper, or silver.

A nanoparticle that allows controlled release of hypochlorous acid oracetic acid permits diffusion of the acid to occur more slowly than theacid would diffuse from an equal volume of the same aqueous solution ofthe acid that is not encapsulated in a nanoparticle. The controlledrelease of hypochlorous acid or acetic acid may be due to permeabilitycharacteristics of the nanoparticle, e.g., a nanoparticle that ispartially or poorly permeable to the acid. A controlled-releasenanoparticle may be a nanoparticle that releases the acid due todegradation of the nanoparticle or impairment of its structuralintegrity in a time-dependent manner. Release of the acid from thenanoparticle may be triggered by environmental conditions, such as pH,temperature, light, pressure, redox conditions, or the presence of aparticular chemical.

FIG. 14 is an illustration of a method 1401 of making an anti-microbialcomposition that includes an aqueous solution 1403 of hypochlorous acidencapsulated in a nanoparticle 1405. The method entails mixing 1411 inwater in a chamber 1413 from which air has been purged a compound 1415that generates a proton (H⁺) in water and a compound 1417 that generatesa hypochlorite anion (OCl⁻) in water. The mixing 1411 produces anair-free aqueous solution 1403 of hypochlorous acid. The solution 1403is then encapsulated 1421 in a nanoparticle 1405. The encapsulation maybe performed in an air-free environment to produce a composition that issubstantially free of air.

INCORPORATION BY REFERENCE

Any and all references and citations to other documents, such aspatents, patent applications, patent publications, journals, books,papers, web contents, that have been made throughout this disclosure arehereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

EXAMPLES Example 1 Product Analysis

When spectrophotometry is expanded to also cover the visible range it ispossible to detect colors. The gases generally produced duringproduction of HOCl are ClO₂, ClO₂O and Cl₂, all of which are detectablein the visible range as yellow or yellow-red. Tzanavaras et al. (CentralEuropean J. of Chemistry, 2007, 5(1)1-12). Data in FIG. 9 illustratesthat the HOCl produced by methods on the invention shows no absorptionfrom colored gases as shown by the lack of colored substance. It isknown that HOCl produces a peak at 292 nm (Feng et al. 2007, J. Environ.Eng. Sci. 6, 277-284).

Example 2

HOCl produced by the process described above was stored under heatstress at 40° C. in order to accelerate degradation using four differenttypes of aqueous solutions: (1) reagent grade water (deionized water);(2) tap water; (3) reagent grade water with a phosphate buffer; and (4)tap water with a phosphate buffer. Characteristics of the HOCl productwere monitored after the initial reaction (T=0); four weeks (T=4); eightweeks (T=8); and twelve weeks (T=12).

FIG. 10 is a graph showing the amount (parts per million (ppm)) of HOClinitially produced (T=0) and its stability over time. The data show thatthe reagent grade water (deionized water) without phosphate buffer isthe most stable over the twelve weeks, showing the least amount ofproduct degradation from the initial amount produced. The deionizedwater produces a much more stable product than that produced using tapwater. Additionally, and surprisingly, the data show that phosphatebuffer may negatively impact amount of HOCl product produced.

FIG. 11 is a graph showing how the pH of the HOCl product changed overtime. In all cases, the pH decreased over time, however, for all cases,the pH stayed in the range of pH=4 to pH=7 over the twelve weeks.

FIG. 12 is a graph showing the oxidation capacity of the HOCl productover time. The data show that the product retained oxidation capacityover the twelve weeks regardless of the starting water.

Example 3 Acetic Acid Compared to Hydrochloric Acid

Using the above described process, HOCl was produced using hydrochloricacid (HCl) and acetic acid and thereafter stored under heat stress at 40C. The amount of HOCl initially produced was recorded (T=0) and then theamount of HOCl product remaining after twelve days was recorded. Threebatches of each were produced. The data for the HCl produced HOCl isshown in Table 1. The data for the acetic acid produced HOCl is shown inTable 2.

TABLE 1 HOCl produced with HCl Initial Amount Amount Batch amountInitial after 12 days pH after Amount of pH number (ppm) pH (ppm) 12days degradation change 1 192 7.12 159 5.71 17.2% 19.8% 2 183 5.88 1474.01 19.7% 31.8% 3 189 5.21 154 3.97 18.5% 23.8%

TABLE 2 HOCl produced with acetic acid Initial Amount Amount Batchamount Initial after 12 days pH after Amount of pH number (ppm) pH (ppm)12 days degradation change 1 205 4.62 180 4.50 12.4% 2.7% 2 205 5.33 1785.04 13.3% 5.4% 3 207 4.07 178 3.89 13.9% 4.6%

The data show that using acetic acid provides greater product stability,most likely due to greater stability in the pH. Without being limited byany particular theory or mechanism of action, it is believed that thedifferent protonation capacity of acetic acid as compared tohydrochloric acid, i.e., acetic acid donates fewer protons to a liquidthan hydrochloric acid, results in greater HOCl stability over time.

What is claimed is:
 1. A wound dressing comprising: a multi-layeredsubstrate comprising at least a wound-contacting surface layer that isformed from a material selected from the group consisting of a naturalfiber, alginate, chitosan, chitosan derivatives, cellulose,carboxymethyl-cellulose, cotton, rayon, nylon, acrylic, polyester,polyurethane foam, hydrogels, hydrocolloids, polyvinyl alcohol, starch,a starch film, collagen, hyaluronic acid and its derivatives, andbiodegradable materials; and a dosage of a composition for treating awound provided on the wound-contacting surface layer of the substrate,the composition comprising an acetic acid component and a hypochlorousacid component and being produced and stored in an environmentsubstantially free of air such that the composition is substantiallyfree of air, wherein a mass concentration of the acetic acid componentin the composition is greater than 0.1% and a concentration of thehypochlorous acid component in the composition is between 10 ppm and1000 ppm such that, upon application of the composition to a wound, thehypochlorous acid component is effective at treating biofilm on asurface or just below the surface of the wound and the acetic acid iseffective at penetrating the surface of the wound to thereby allowdeeper, sub-surface treatment of biofilm infected tissue; and whereinthe acetic acid component is encapsulated in a nanoparticle.
 2. Thecomposition of claim 1, wherein the mass concentration of the aceticacid component is greater than about 0.5%.
 3. The composition of claim2, wherein the mass concentration of the acetic acid component isgreater than about 1.0%.
 4. The composition of claim 3, wherein the massconcentration of the acetic acid component is greater than about 2.0%.5. The composition of claim 1, wherein the hypochlorous acid componentconcentration is between 20 ppm and 200 ppm.
 6. The composition of claim1, wherein the composition is formulated in a gel, cream, ointment, oroil.